Circular RNA cFAM210A, degradable by HBx, inhibits HCC tumorigenesis by suppressing YBX1 transactivation

Hepatitis B protein x (HBx) has been reported to promote tumorigenesis in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC), but the mechanism awaits further investigation. In this study, we found that cFAM210A (a circular RNA derived from the third exon of transcript NM_001098801 of the FAM210A gene; CircBase ID: hsa_circ_0003979) can be silenced by HBx. cFAM210A expression was downregulated and negatively correlated with tumorigenesis in patients with HBV-related HCC. Furthermore, cFAM210A reduced the proliferation, stemness, and tumorigenicity of HCC cells. Mechanistically, HBx increased the N6-methyladenosine (m6A) level of cFAM210A by promoting the expression of RBM15 (an m6A methyltransferase), thus inducing the degradation of cFAM210A via the YTHDF2-HRSP12-RNase P/MRP pathway. cFAM210A bound to YBX1 and inhibited its phosphorylation, suppressing its transactivation function toward MET. These findings suggest the important role of circular RNAs in HBx-induced hepatocarcinogenesis and identify cFAM210A a potential target in the prevention and treatment of HBV-related HCC.


Follow-up
The patients of cohort 3 received check-ups every 2-3 months after surgery during the first 24 months and every 3-6 months thereafter until November 10, 2016.The median follow-up period was 45.2 months.
Physicians who were blinded to the study performed the follow-up examinations.Serum AFP levels and abdominal ultrasound examinations were performed every month during the first year after surgery and every 3-6 months thereafter.Computed tomography and/or magnetic resonance imaging were performed every 3-6 months or when a recurrence was suspected.The diagnosis of recurrence was based on the diagnosis criteria from the AASLD Practice Guidelines (http://www.aasld.org/practiceguidelines/Documents/Bookmarked%20Practice%20Guidelines/HCCUpdate2010.pdf)Once recurrence was confirmed, further treatment was implemented based on the tumor diameter, the number of tumors, the location of the tumor, and the extent of vessel invasion as well as liver function and performance statuses.Recurrence-free survival (RFS) was calculated from the date of tumor resection until the detection of tumor recurrence, death from a cause other than HCC, or the last follow-up visit.

Whole transcriptome sequencing and identification of differentially expressed circRNAs
Total RNA was extracted from HBx-overexpressing (HBx-oe) and NC (negative control) HepG2 cells using Trizol reagent (Invitrogen, CA) following the manufacturer's protocol.The RNA concentration and quality were evaluated with the NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies).
Firstly, rRNA was depleted by NEBNext rRNA Depletion Kit(New England Biolabs, Inc., Massachusetts, USA)according to the manufacturer's protocol.Secondly, sequencing libraries were prepared using NEBNext ® Ultra™ II Directional RNA Library Prep Kit(New England Biolabs, Inc., Massachusetts, USA).BioAnalyzer 2100 system(Agilent Technologies, USA)was used in quality control.
The libraries were sequenced on an Illumina HiSeq 4000 platform (Illumina, San Diego, CA, USA) according to the manufacturer's instructions, and 150-bp paired-end reads were generated.Cutadapt (V1.9.3) 1 was used to acquire high-quality reads.
The high-quality reads were then mapped to the Homo sapiens Hg19 genome using STAR (V2.5.1b) 2 .

Quantitative real-time PCR
Total RNAs were extracted using Trizol reagent (Invitrogen, CA).The first-strand cDNA was generated using the M-MLV Reverse Transcriptase kit (Invitrogen, CA) with random primers.Real-time PCR reactions were performed in the StepOne TM Real-Time PCR System (Applied Biosystems, Foster City, CA).
The real-time PCR reactions were performed in triplicate.ACTB were employed as endogenous control for mRNA and miRNAs, respectively.The relative expression was calculated using the comparative ΔΔCt method.The primer sequences are presented in Supplementary Table 6.

Transient transfection
The transient transfection of small interfering RNAs and plasmids were performed using the Lipofectamine 3000 kit (Invitrogen) according to the manufacturer's instructions.The siRNA sequences are listed in Supplementary Table 6.

Western blot analysis:
Total protein was extracted from snap-frozen tissues with RIPA Lysis Buffer and PMSF (Beyotime Co., China) according to the manufacturer's instructions.Western blotting was performed as described previously 7 .Antibody binding was detected with an Odyssey infrared scanner (Li-Cor Biosciences Inc.).
The antibodies used are listed in Supplementary Table 7.

Cell lines
Hep3B, HepG2, Huh7 and MHCC97H cells were obtained from the Chinese Academy of Sciences Cell Bank and were authenticated by short tandem repeat (STR) profiling.Cells were grown in Dulbecco's modified Eagle's medium with 10% foetal bovine serum (Gibco BRL).Cells were maintained in an atmosphere of 5% CO2 in a humidified 37°C incubator.Primary human hepatocyte (HH) (Lot #M00995-P) was obtained from RILDbiotech (Shanghai, China) and cultured as described previously 8 .

RNA immunoprecipitation.
RNA immunoprecipitation (RIP) experiments were performed using a Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) according to the manufacturer's instructions.

Actinomycin D assay
The Actinomycin D assay was performed as previously described [9][10][11] .HCC cells were equally seeded in 5 wells in 24-well plates (5 × 104 cells per well).24 hours later, the cells were exposed to actinomycin D (2µg/ml, Abcam, ab141058) for 0h, 12h, 24h and 48h, respectively.After that, the cells were harvested and the relative RNA levels of cFAM210A were analyzed by qRT-PCR and normalized to the values measured in the mock treatment group (the 0h group).

Promoter luciferase reporter assay
The promoter of RBM15 and MET were inserted into the vector GV238 (Genechem) respectively.
Dual-luciferase reporter assay was carried out completely in line with the manufacturer's instructions (Beyotime, Shanghai, China).

Dual RNA fluorescence in situ hybridization (FISH) and immunofluorescence assay
CY3-labeled probe (Supplementary Table 5) to cFAM210A back-slice sequence was synthesized by RiboBio (Guangzhou, China).The probe signals were detected by the Fluorescent In Situ Hybridization Kit (RiboBio), following the manufacturer's instructions.HepG2 cells were incubated with antibodies specific for human YBX1 (20339-1-AP, Proteintech Group) at 4℃ overnight and then with FITC-labeled goat anti-rabbit IgG for 30 minutes.Subsequently, the nuclei were re-dyed with DAPI.Images were taken with Nikon Eclipse E200 Microscope (Japan).
Transfected cells were plated onto a 96-well plate at a cell density of 2000 cells per well for 24 h.Next, the viability of the cells was measured at 450 nm using Synergy 2 (BioTek, USA) every 24 h for 4 days.10 μL of CCK8 assay was added 2 hours before measurement.

5-Ethynyl-20-deoxyuridine (EdU) incorporation assays
The EdU assay was carried out with a Cell Light EdU DNA Cell Proliferation Kit (RiboBio, Shanghai, PR, China) according to the protocol as described before. 12Images were acquired with Zeiss axiophot photomicroscope (Carl Zeiss) and Image-Pro plus 6.0 software, and the percentage of EdU-positive cells was calculated.

Sphere formation assays
The spheres formation assay was performed as previously described. 121000 cells per well were seeded in ultra-low adherent-conditioned plates (Corning, USA) with serum-free DMEM medium containing B27 supplement (1:50; Invitrogen), 20 ng/mL epidermal growth factor (Invitrogen) and 20 ng/mL basic fibroblast growth factor (Invitrogen) for 14 days to test their ability of forming primary spheres.On day14, cell sphere number of spheres was counted using an inverted microscope (Olympus, Tokyo, Japan).

In vitro limiting dilution assays
HCC cells were seeded into 96-well ultra-low attachment culture dishes at cell doses described in the body of article and incubated in spheroid-forming conditions for 14 days.Sphere formation was assessed by visual inspection.Based on the frequency of wells without spheroids, the proportion of spheroid-initiating cells was determined using Poisson's distribution statistics and the L-Calcsoftware program (Version 1.1; Stem Cell Technologies, Inc., Vancouver, British Columbia, Canada).

In vivo limiting dilution assays
Male nude mice were purchased from the Laboratory Animal Resources, Chinese Academy of Sciences (Beijing, China) and received humane care.Various amounts of HCC cells were injected subcutaneously of nude mice, as described before. 12Kinetics of tumor formation were estimated twice weekly, and mice were monitored for 8 weeks.Frequency of tumor-initiating cells was determined using Poisson's distribution statistics and the L-Calc software program (Version 1.1; Stem Cell Technologies).